The present invention generally relates to promoters, enhancers and other regulatory elements of smooth muscle cells (xe2x80x9cSMCxe2x80x9d). The invention more particularly relates to methods for the targeted knockout, or over-expression, of genes of interest within smooth muscle cells. The invention further relates to methods of conferring smooth muscle cell specific gene expression in vivo.
Smooth muscle cells, often termed the most primitive type of muscle cell because they most resemble non-muscle cells, are called xe2x80x9csmoothxe2x80x9d because they contain no striations, unlike skeletal and cardiac muscle cells. Smooth muscle cells aggregate to form smooth muscle which constitutes the contractile portion of the stomach, intestine and uterus, the walls of arteries, the ducts of secretory glands and many other regions in which slow and sustained contractions are needed.
Abnormal gene expression in SMC plays a major role in numerous diseases including, but not limited to, atherosclerosis, hypertension, stroke, asthma and multiple gastrointestinal, urogenitcl and reproductive disorders. These diseases are the leading causes of morbidity and mortality in Western Societies, and account for billions of dollars in health care costs in the United States alone each year.
In recent years, the understanding of muscle differentiation has been enhanced greatly with the identification of several key cis-elements and trans-factors that regulate expression of muscle-specific genes. Firulli A. B. et al., 1997, Trends in Genetics, 13:364-369; Sartorelli V. et al., 1993, Circ. Res., 72:925-931. However, the elucidation of transcriptional pathways that govern muscle differentiation has been restricted primarily to skeletal and cardiac muscle. Currently, no transcription factors have yet been identified that direct smooth muscle-specific gene expression, or SMC myogenesis. Owens G. K., 1995, Physiol. Rev., 75:487-517. Unlike skeletal and cardiac myocytes, SMC do not undergo terminal differentiation. Furthermore, they exhibit a high degree of phenotypic plasticity, both in culture and in vivo. Owens G. K., 1995, Physiol. Rev., 75:487-517; Schwartz S. M. et al., 1990, Physiol. Rev., 70:1177-1209. Phenotypic plasticity is particularly striking when SMC located in the media of normal vessels are compared to SMC located in intimal lesions resulting from vascular injury or artherosclerotic disease. Schwartz S. M., 1990, Physiol. Rev., 70:1177-1209; Ross R., 1993, Nature, 362:801-809; Kocher 0. et al., 1991, Lab. Invest., 65:459-470; Kocher 0. et al., 1986, Hum. Pathol., 17:875-880. Major modifications include decreased expression of smooth muscle isoforms of contractile proteins, altered growth regulatory properties, increased matrix production, abnormal lipid metabolism and decreased contractility. Owens G. K., 1995, Physiol. Rev., 75:487-517. The process by which SMC undergo such changes is referred to as xe2x80x9cphenotypic modulationxe2x80x9d. Chamley-Campbell J. H. et al., 1981, Atherosclerosis, 40:347-357. Importantly, these alterations in expression patterns of SMC protein cannot simply be viewed as a consequence of vascular disease, but rather are likely to contribute to progression of the disease.
A key to understanding SMC differentiation is to identify transcriptional mechanisms that control expression of genes that are selective or specific for differentiated SMC and that are required for its principal differentiated function, contraction. Currently, studies are ongoing in which the expression of the contractile proteins SM xcex1-actin (Shimizu R. T. et al., 1995, J. Biol. Chem., 270:7631-7643; Blank R. S. et al., 1992, J. Biol. Chem., 267:984-989) and SM myosin heavy chain (SM-MHC)(White S. L. et al., 1996, J. Biol. Chem., 271:15008-15017; Katoh Y. et al., 1994, J. Biol. Chem., 269:30538-30545; Wantanabe M. et al., 1996, Circ. Res., 78:978-989; Kallmeier R. C. et al., 1995, J. Biol. Chem., 270:30949-30957; Madsen C. S. et al., 1997, J. Biol. Chem., 272:6332-6340; Madsen C. S. et al., 1997, J. Biol. Chem., 272:29842-29851), as well as a variety of proteins implicated in control of contraction including SM22xcex1 (Li L. et al., 1996, J. Cell. Biol., 132:849-859; Kim S. et al., 1997, Mol. Cell. Biol., 17:2266-2278), h1-calponin (Miano J. M. et al., 1996, J. Biol. Chem., 271:7095-7103), h-caldesmon (Yano H. et al., 1994, Biochem. Biophys. Res. Commun., 201:618-626), telokin (Herring B. P. et al., 1996, Am. J. Physiol., 270:C1656-C1665) and desmin (Bolmont C. et al., 1990, J. Submicrosc. Cytol. Pathol., 22: 117-122) are being examined. Of these gene products, only SM-MHC expression appears to be completely restricted to SMC lineages throughout development (Miano J. et al., 1994, Circ. Res., 75:803-812), whereas all others show at least transient expression in non-SMC tissues (Owens G. K., 1995, Physiol. Rev., 75:487-517). As such, it appears that the SM-MHC gene is unique with regard to its potential utility for identification of SMC-specific transcriptional regulatory pathways and mechanisms.
To date, four SM-MHC isoforms (SMC-1A, SMC-1B, SMC-2A and SMC-2B) have been identified (Nagai R. et al., 1989, J. Biol. Chem., 264:9734-9737; White S. et al., 1993, Am. J. Physiol., 264:C1252-C1258; Kelley C. A. et al., 1993, J. Biol. Chem., 268:12848-12854), all of which are derived from alternative splicing of a single gene (Miano J. et al. 1994, Circ. Res., 75:803-812; Babij P. et al., 1989, J. Mol. Biol., 210:673-679). Alterations in expression of SM-MHC isoforms have been extensively documented in SMC that have undergone phenotypic modulation either when placed in culture (Rovner A. S., 1986, J. Biol. Chem., 261:14740-14745; Kawamoto S. et al., 1987, J. Biol. Chem., 262:7282-7288), or in vascular lesions of both humans and several animal models of vascular disease (Aikawa M. et al., 1997, Circulation, 96:82-90; Sartore S, et al., 1994, J. Vasc. Res., 31:61-81). Thus, the SM-MHC gene represents an excellent candidate gene for delineating transcriptional pathways important for both normal development and diseased states.
Transcriptional regulation of the SM-MHC gene has been analyzed extensively in cultured SMC and several functional cis-elements have been identified. White S. L. et al., 1996, J. Biol. Chem., 271:15008-15017; Katoh Y. et al., 1994, J. Biol. Chem., 269:30538-30545; Wantanabe M. et al., 1996, Circ. Res., 78:978-989; Kallmeier R. C. et al., 1995, J. Biol. Chem., 270:30949-30957; Madsen C. S. et al., 1997, J. Biol. Chem., 272:6332-6340; Madsen C. S. et al., 1997, J. Biol. Chem., 272:29842-29851. However, because differentiation of SMC is known to be dependent on many local environmental cues that cannot be completely reproduced in vitro, cultured SMC are known to be phenotypically modified as compared to their in vivo counterparts (Owens G. K., 1995, Physiol. Rev., 75:487-517; Chamley-Campbell J. H. et al., 1981, Atherosclerosis, 40:347-357). As such, certain limitations may apply regarding the usefulness of cultured SMC in defining transcriptional programs that occur during normal SMC differentiation and maturation within the animal.
Prior to the instant invention, no genetic elements that are completely specific for SMC and which have been proven to confer smooth muscle specific gene expression in vivo in transgenic animals have been defined, isolated or identified. Furthermore, as discussed above, previously characterized smooth muscle cell gene promoters including those for SM 22xcex1 and SM xcex1-actin show activity in both SMC and non-SMC, thus limiting their use for purposes requiring SMC-specific gene targeting.
The current invention provides the major advance of identifying molecular elements that confer SMC-specific transcription in vivo during normal development. More specifically, the instant invention utilizes transgenic mice to identify DNA sequences that are critical for SM-MHC expression. Thus, the instant invention provides, for the first time, the identification of sufficient regions of the SM-MHC gene to direct SMC-specific expression both in vitro in cultured SMC and in vivo in transgenic mice. Therefore, the instant invention can be used, for example, for the targeted knockout, or over-expression, of genes of interest within smooth muscle cells. Potential applications for the instant invention include, for example, the treatment or possible cure of the many diseases involving smooth muscles, including, but not limited to, coronary artery disease, asthma and hypertension.
The present invention generally relates to promoters, enhancers and other regulatory elements of genes. More particularly, the invention is directed to regulatory elements that confer SMC-specific gene expression both in vitro and in vivo.
One aspect of the invention relates to the use of SM-MHC promoters and other regulatory elements to control the expression of protein and RNA products in SMC. SM-MHC promoters and other regulatory elements have a variety of uses including, but not limited to, expressing heterologous genes in SMC tissues, such as the contractile portion of the stomach, intestine and uterus, the walls of arteries, the ducts of secretory glands and many other regions in which slow and sustained contractions are needed.
Another aspect of the invention relates to the use of SM-MHC promoters and other regulatory elements for genetic engineering as a means to investigate SMC physiology and pathophysiology. For example, a specific gene that is believed to be important for a specific disease within SMC could be knocked out with the confounding influences of knocking out that gene in other cell types and tissues. This could be accomplished by methods well known to those of skill in the art. For example, an antisense polynucleotide could be expressed under the control of an SM-MHC that would inhibit a target gene of interest, or an inhibitor could be expressed that would specifically inhibit a particular protein.
In an alternative embodiment of the invention, the SM-MHC promoter/enhancer is used to carry out targeted knockout of genes of interest. For example, a number of tetracycline-cre-recombinase based mouse systems can be used to obtain SMC targeting of cre-recombinase dependent genes (i.e. xe2x80x9cfloxedxe2x80x9d genes containing lox p cre recombinase recognition sites) of interest. Further, one could examine how selective (SMC-specific) knockout of an SMC gene of interest affects development of coronary artery disease without the confounding limitations of conventional knockouts with respect to deducing the primary site of action, activation of compensatory pathways, etc. The feasibility of these sorts of approaches has been shown in other, non-SMC, tissue types (see, Mayford et al., Science 274:1678, 1996). However, the invention described herein discloses, for the first time, such studies in SMC tissues. For example, the SM-MHC of the instant invention can be used in combination with the tetracycline-cre-recombinase based mouse systems to effectuate targeted knockouts of various genes which are implicated in the control of SMC differentiation within SMC tissues. (Hautmann et al. Circ. Res. 81:600,1997; Blank et al., Circ. Res. 76:742, 1995; Madsen et al, J. Biol. Chem. 272:6332,1997, each of which is incorporated by reference in its entirety). Examples of such genes include genes which encode for serum response factor, the homeodomain protein MHox and the retinoic acid xcex1-receptor. It is of interest that conventional (non-targeted) knockout of these genes results in embryonic lethality, thus precluding the utility of studying involvement of these genes in control of SMC differentiation in diseases such as atherosclerosis, hypertension, asthma, etc.
A major biomedical application of the invention would be to use the SM-MHC regulatory region to over-express a gene of interest within SMC. For example, an inhibitor of a pathologic process within an SMC tissue may be over-expressed in order to generate a high, local concentration of the factor that might be needed for a therapeutic effect. Since expression of the gene would be SMC-specific, undesired side effects on other tissues that often result when conventional systemic administration of therapeutic agents are utilized would be avoided. For example, a gene for an SMC relaxant could be over-expressed within bronchiolar SMC as a therapy for asthma, or an inhibitor of SMC growth could be over-expressed to prevent development of atherosclerosis or post-angioplasty restinosis. As shown in FIG. 6, the SM-MHC transgene of the instant invention was specifically expressed at high levels within all coronary arteries and arterioles within the heart of an adult mouse, thus demonstrating the efficacy of the SM-MHC promoter/enhancer for gene therapy for coronary artery disease.
The present invention is based, in part, on the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle-specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-lacZ reporter construct containing approximately 16 kb of the SM-MHC genomic region from about xe2x88x924.2 kb to about +11.7 kg (within the first intron) expressed the lacZ transgene in all smooth muscle tissue types. The inclusion of intronic sequence was required for transgene expression since 4.2 kb of the 5xe2x80x2 flanking region alone was not sufficient for expression.
Furthermore, in the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, airway smooth muscle of the trachea and bronchi and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters and bladder. In addition, of particular significance, the transgene was expressed at high levels throughout the coronary circulation. (See, FIG. 6). During development, transgene expression was first detected in airway SMC at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5.
Thus, the present invention discloses for the first time, a promoter/enhancer region of SM-MHC that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory elements, and to design vectors for SMC-specific gene targeting.